We previously identified a new class of nucleoside reverse transcriptase inhibitors (NRTIs), 4'-ethynyl-2'-deoxunucleoside analogs (EdNs), which, unlike conventional NRTIs such as azidothymidine, contain the 3'-hydroxyl moiety. These EdNs exert potent antiviral activity against a wide spectrum of HIV-1 and HIV-2 strains. In the time period of this annual report, we further examined the in vivo activity of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), one of EdNs, which has a potent activity against various HIV-1 and simian immunodeficiency virus (SIV) strains including multi-drug resistant HIV-1 variants and is thought to be suitable for once daily regimen, in two SIV-infected monkeys, in collaboration with Dr. Michael Parniak of University of Pittsburgh. EFdA treatment resulted in a 2-3 log decrease in SIV load within 7 days. Despite the 3'-hydroxyl group, EFdA acts as a chain terminator by preventing RT translocation. The 4'-ethynyl group locks the sugar in a favorable position for incorporation, which along with the 3'-hydroxyl makes EFdA a better substrate for RT than native nucleotides. Similarly, EFdA may be preferred by Pol gamma, but kinetic studies are limited to an IC50 value of 10 microM for Pol gamma, indicating EFdA can serve as a substrate. In this project, in collaboration with Dr. Karen Anderson of Yale University, we expanded our prior work with EFdA to characterize the molecular mechanism of inhibition of Pol gamma by EFdA-TP. Such studies are critical to possibly assess the safety of drugs in preclinical and clinical trials. Pol gamma incorporated EFdA-TP 760-fold more slowly and with 5.6-fold lower affinity vs dATP. The efficiency of EFdA-TP incorporation is well within the range of NRTIs on the market, showing 340- and 1.5-fold improvement over stavudine (D4T-TP) and didanosine (ddA-TP), respectively. Although lower efficiencies are seen with tenofovir (3.2-fold) and AZT (16-fold), it is important that EFdA in the steady-state showed 440-fold and 66,000-fold higher potency than AZT and tenofovir, respectively. More importantly, in contrast to RT, Pol gamma shows a 4,300-fold preference for dATP over EFdA-TP, and this coupled with the very slow incorporation rate and low affinity for EFdA-TP indicates a very low risk of Pol gamma- mediated toxicity. It is of note that when we propagated HIV-1 in the presence of increasing concentrations of EFdA, a moderately EFdA-resistant HIV-1 variant population was selected after as many as 58 passages (the susceptibility fold-difference of 24), which contained a unique combination of mutations, I142V/T165R/M184V. Analyses using newly generated recombinant infectious clones with site-directed mutagenesis showed that M184V was the main mutation responsible for EFdA resistance, and the addition of I142V and/or T165R augments the effect of M184V. Nitanda and his colleagues have identified that a similar HIV-1 variant was selected when HIV-1 was propagated in the presence of an EFdA congener, 4'-ethynyl-2',3'-didehydro-dideoxythymidine (Ed4T). Most recently we compared the antiretroviral potency of EFdA and Ed4T head-to-head using MT4 cells as target cells and found that the IC50 values for EFdA and Ed4T were 2 nM and 200 nM, respectively. When HIV-1 was propagated in the presence of EFdA or Ed4T, Ed4T-resistance was relatively readily identified compared to EFdA-resistance (Unpublished data by Maeda and Mitsuya). These data, together, warrant that EFdA be further developed toward clinical application. In this project, we designed and synthesized novel oxatricyclic [3(R), 3aS, 4aS, 6aR, 7aS] and [3(R), 3aS, 4aR, 6aS, 7aS] ligands and incorporated these ligands in the (R)-hydroxyethyl sulfonamide isostere. The orientation, ring size, and stereochemistry are all critical to the potency of the oxatricyclic ligand-derived inhibitors. The ligands were synthesized with defined stereochemistry in optically active forms. Incorporation of a syn-syn-syn-fused tris-THF led to a prototype to GRL-0519A, which has significantly decreased enzyme inhibitory and antiviral potency relative to DRV. However, the tris-THF ligand with a syn-anti-syn fusion provided inhibitor GRL-0519A, which has remarkable enzyme inhibitory and antiviral activity. Of particular interest, GRL-0519A displayed potent activity against a variety of multidrug-resistant clinical HIV-1 strains, with EC50 values ranging from 0.6 to 4.3 nm, a nearly 10-fold improvement over DRV. Moreover, GRL-0519A blocked protease dimerization at least 10-fold more potently than DRV. An X-ray crystal structure of GRL-0519A-bound HIV-1 protease revealed molecular insight into the ligand-binding site interactions responsible for its potency against wild-type and mutant viruses. It appears that the first and second THF rings of the tris-THF ligand witha syn-anti-syn configuration maintain key backbone hydrogen bonding interactions similar to the bis-THF ligand of darunavir. The third THF ring oxygen makes water-mediated hydrogen bonds with Asp29, Arg8', and Gly27. In addition, the ring fills the hydrophobic pocket in the S2 subsite and also stacks nicely behind the P1 phenylmethyl substituent at the S1 subsite. Therefore, our basic design strategy has shown to be extremely powerful and is well worth further experimentation. In this project, we also identified GRL-1388 and -1398, potent non-peptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a bicyclic P2 functional moiety, tetrahydropyrano-tetrahydrofuran (Tp-THF). GRL-1388 was as potent as darunavir (DRV) against various drug-resistant HIV-1 laboratory strains with EC50 values of 2.6 to 32.6 nM. GRL-1398 was significantly more potent against such variants than DRV with EC50 values of 0.1 to 5.7 nM. GRL-1388 and -1398 were also potent against multiple-PI-resistant clinical HIV-1 variants (CLHIV-1MDR) with EC50s ranging 2.7-21.3 nM and 0.3-4.8 nM, respectively. A highly DRV-resistant HIV-1 variant selected in vitro remained susceptible to GRL-1398 with the EC50 of 21.9 nM, while the EC50 value of DRV was 214.1 nM. When HIV-1NL4-3 was selected with GRL-1398, four amino acid substitutions, leucine to phenylalanine at a position 10 (L10F), A28S, L33F and M46I emerged, ultimately enabling the virus to replicate in the presence of &gt;1.0 microM of the compound beyond 57 weeks of selection. When a mixture of 10 different CLHIV-1MDR was selected, the emergence of resistant variants was substantially delayed with GRL-1398 than with GRL-1388 and DRV. Modeling analyses revealed that GRL-1398 had greater overall hydrogen bonding and hydrophobic interactions than GRL-1388 and DRV and that GRL-1388 and -1398 had hydrogen bonding interactions with the main chain of the active site amino acids (Asp29 and Asp30) of protease. The present findings warrant that GRL-1398 be further developed as a potential drug for treating individuals with HIV-1 infection. Moreover, in this project, we have designed and synthesized a stereochemically defined hexahydrofuropyranol-derived urethane as the P2-ligand. The current ligand is designed based upon X-ray structure of darunavir-bound HIV-1 protease. The synthesis of (3aS,4S,7aR)-hexahydro-2H-furo[2,3-b] pyran-4-ol was carried out. Incorporation of this ligand provided inhibitor GRL-0476, which has shown excellent enzyme inhibitory activity and antiviral potency. Our structure activity studies have indicated that the stereochemistry and the position of oxygens in the ligand are important to the observed potency of the inhibitors. GRL-0476 maintains excellent potency against multidrug-resistant HIV-1 variants. An active site model of GRL-0476 was created based upon the X-ray structure of HIV-1 protease. The model offers molecular insights regarding ligand-binding site interactions of the hexahydrofuropyranol-derived novel P2-ligands.